Method for inducing a systemic immune response to an HIV antigen

ABSTRACT

A method is provided for inducing a systemic immune response to an antigen selected from inactivated HIV I and HIV II antigens in a mammal. The method comprises orally administering lyophilized multilaminar liposomes containing the antigen. The liposomes have a size of from 20 nm to 20 microns. The antigen-containing liposomes are absorbed in the Peyer&#39;s patches of the gut. Sufficient antigen-containing liposomes are taken up by macrophages in the Peyer&#39;s patches to induce a systemic immune response to the antigen.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/882,968, filed Jun. 26, 1997, which is a continuation-in-part ofInternational applicatin No. PCT/US97/04634, filed Mar. 24, 1997, whichis a continuation of U.S. application Ser. No. 08/621,802, filed Mar.22, 1996, now abandoned; this application is also a continuation-in-partof U.S. application Ser. No. 08/920,374, filed Aug. 29, 1997, which is acontinuation of International application No. PCT/US97/04634, filed Mar.24, 1997, which is a continuation-in-part of U.S. application Ser. No.08/621,802, filed Mar. 22, 1996, now abandoned.

FIELD OF THE INVENTION

This invention relates to a method for inducing a systemic immuneresponse to an HIV antigen and more particularly to vaccines suitablefor oral administration.

BACKGROUND OF THE INVENTION

The epithelial surfaces of the body serve as a barrier to antigenicmaterial. However, those surfaces are by no means impenetrable. Themucosal immune system provides the next major line of defense against amajority of human pathogens. The mucosal immune system includesgut-associated lymphoid tissue (GALT), bronchus-associated lymphoidtissue, the salivary glands, the conjunctiva, the mammary gland, partsof the urogenital tract, and the middle ear.

GALT consists of two types of lymphoid aggregates. The first is referredto as Peyer's patches and the second consists of isolated lymphoidfollicles. Peyer's patches have a defined micro-structure including acentral B cell dependent follicle and T cell dependent regions adjacentto the follicle. The lymphocytes in Peyer's patches are heterogeneous,including B cells which express IgM, IgG, IgA, and IgE and variousregulatory and cytotoxic T cells. Peyer's patches also containspecialized macrophages. The Peyer's patches are covered by M cells,which are specialized lympho-epithelium cells.

In GALT, ingested antigens produce a local immune response. The antigensare taken up by the M cells, which deliver the antigen to the underlyinglymphocytes in the tissue. This results in the production of IgA atvarious secretory effector sites following the migration of activatedlymphocytes through the efferent, lymphatic and circulatory system.

The absorption of antigens by the Peyer's patches can induce a systemicimmune response if the antigen is taken up by macrophages in the Peyer'spatches. Macrophages induce a systemic response by processing antigensand presenting them to lymphocytes. The lymphocytes then becomeactivated and cause the production of systemic antibodies specific tothe antigens.

Childers et al. (Oral Microbiol. Immunol. 1994:9:146-153) reported thatlyophilized liposomes containing S. mutans antigen can be administeredorally to human patients and will be absorbed by GALT to elicit a localimmune response. No systemic response was observed however.

Traditionally, to obtain a systemic immune response by oraladministration of an antigen, it was required that the antigen beassociated with an adjuvant. The presence of the adjuvant permits theantigen/adjuvant combination to be recognized by the CD4 cells, whichsend signals to B cells to produce antibodies, and by the cytotoxiclymphocytes, which kill the infecting organism in affected host cells.Without the presence of the adjuvant, the CD4 cells and cytotoxiclymphocytes ignore the free antigen.

Typical adjuvants include alum, Freund's adjuvant, incomplete Freund'sadjuvant and indotoxin. These adjuvants typically induce an inflammatoryresponse. Other typical adjuvants are immuno stimulating complexes(iscoms) that contain Quil A. These adjuvants typically cause clumpingof antigens.

A need therefore exists to induce a systemic immune response by oraladministration without the presence of an adjuvant and without inducingthe above-described adjuvant effects, but instead by uptake by themacrophages in the Peyer's patches.

SUMMARY OF THE INVENTION

The present invention provides a method for inducing a systemic immuneresponse to one or more antigens in a mammal and does not require thepresence of an adjuvant. According to the present invention, thelyophilized antigen-containing liposomes do not directly target the CD4cells and cytotoxic lymphocytes, but instead are taken up by themacrophages in the Peyer's patches. The macrophages express the antigenin conjunction with self major histocompatibility antigen I and II (SMHI and SMH II). The CD4 cells recognize the antigen expressed with SMH I,and the cytotoxic lymphocytes recognize the antigen expressed with SMHII. Accordingly, the present methods involve an intermediate step, beingtaken up by the macrophages, which is different from the process thatoccurs when an antigen/adjuvant combination is orally administered.Thus, the present invention involves a method whereby the antigencontaining liposomes can be orally administered without an adjuvant toinduce a systemic immune response. Moreover, the inventive methods donot generate an adjuvant effect, e.g., an inflammatory response orclumping of antigens.

The inventive method comprises first incorporating at least one antigenselected from inactivated HIV I and HIV II antigens into liposomes,preferably multilamellar liposomes having a size from about 20 nm toabout 20 microns or greater, preferably from about 200 nm to about 10microns and more preferably from about 1 micron to about 5 microns. Theantigen-containing liposomes are then lyophilized and packaged in asuitable form, such as a pill or capsule, for oral ingestion. Means,such as an enteric coating are provided for preventing breakdown of thepreparation in the stomach but allowing digestion in the gut, i.e.,small intestine. Once orally ingested, the preparation passes throughthe stomach into the gut wherein antigen-containing liposomes areabsorbed in the Peyer's patches of the gut. In the Peyer's patches,sufficient antigen-containing liposomes are taken up by macrophages toinduce a systemic immune response and preferably a long-term systemicimmune response to the antigen(s).

The invention further provides a preparation suitable for oral ingestionfor inducing a systemic response, and preferably a long-term systemicimmune response, to one or more antigens selected from inactivated HIV Iand HIV II antigens. The composition comprises lyophilized, preferablymultilamellar, liposomes that contain the antigen(s). The liposomes havea size, before lyophilization, of from about 20 nm to about 20 micronsor greater, preferably from about 200 nm to about 10 microns, and morepreferably from about one to about five microns. A particularlypreferred composition comprises liposomes of varying sizes includingsmall liposomes, i.e., about 20 mn to about 1 micron, medium liposomes,i.e., about 1 to about 3 microns, and large liposomes, i.e. about 3 toabout 20 microns or greater and preferably about 3 to about 5 microns.It is presently preferred that such a composition comprise at least 5%by volume small liposomes, at least 10% by volume medium liposomes, andat least 20% by volume large liposomes. The composition preferablycomprises means for preventing breakdown of the preparation in thestomach but for allowing digestion of the liposomes in the gut. In thegut, the liposomes are absorbed by Peyer's patches and sufficientliposomes are taken up by macrophages to stimulate a long term systemicimmune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electron photomicrograph (magnification: 100,000×) ofliposomes in lymphoid tissue of a Peyer's patch.

FIG. 2 is an electron photomicrograph (magnification: 10,000×) oflymphoid tissue within the Peyer's patch.

FIG. 3 is an electron photomicrograph (magnification: 20,000×) ofsplenic lymphoid cells.

FIG. 4 is an electron photomicrograph (magnification: 60,000×) ofsplenic lymphoid cells.

FIG. 5 is an electron photomicrograph (magnification: 15,000×) of amacrophage in the Peyer's patch.

FIG. 6 is an electron photomicrograph (magnification: 10,000×) of anextracellular space in the Peyer's patches.

FIG. 7 is an electron photomicrograph (magnification: 15,000×) of anextracellular space in the Peyer's patches.

FIG. 8 is an electron photomicrograph (magnification: 10,000×) ofliposomes surrounding a white blood cell in a venule of the Peyer'spatch.

FIG. 9 is an electron photomicrograph (magnification: 50,000×) of thecytoplasm and cellular membrane of a macrophage in the Peyer's patch.

FIG. 10 is an electron photomicrograph (magnification: 40,000×) showingliposomes at the cellular membrane and inside a macrophage in thePeyer's patch.

FIG. 11 is an electron photomicrograph (magnification: 70,000×) showingliposomes inside a macrophage in the Peyer's patch.

FIG. 12 is an electron photomicrograph (magnification: 75,000×) showingliposomes adhering to a venule wall in the lymphoid cells of the Peyer'spatch.

FIG. 13 is an electron photomicrograph (magnification 25,000×) showing980 nm liposomes in a macrophage vacuole 7 days after oral inoculation.

FIG. 14 is an electron photomicrograph (magnification 12,500×) showing10 micron liposomes in a macrophage 21 days after oral inoculation.

FIG. 15 is an electron photomicrograph (magnification 40,000×) showing 2micron liposomes in a macrophage vacuole 60 days after oral inoculation.

DETAILED DESCRIPTION

Some antigens require intracellular processing by antigen processingcells, such as macrophages or Kupffer cells before being presented to Tlymphocytes as a processed antigen. This processed antigen is thendisplayed on the macrophage surface in association with HLA moleculesand presented to the T cell to confer systemic immunity. The macrophagealso produces certain soluble cytokines that have an important role inT-cell activation, which confers systemic immunity as well.

Therefore, it is critical that the presenting antigen, such as liposomallyophilized antigen, enter the macrophage of the GALT for processing toconfer systemic immunity, and this is dependent upon the size of theliposome presented to the GALT.

Size and composition of the liposomes are important in determining theduration of the systemic immune response to the incoiporated antigen.Administration of liposomes of varying size and composition ensure along lasting immune response, and thus avoid the need for repeatedvaccine administrations. Since the half life of the macrophage isapproximately 90 days, the presentation of an antigen taken up by GALTmacrophages can last up to 180 days for conferring systemic immunity.

It has been found that liposomes containing one or more antigens andhaving a particular size from about 20 nm to about 20 microns orgreater, preferably from about 200 nm to about 10 microns and morepreferably from about 1 to about 5 microns, when administered orally toa mammal in lyophilized form, will be absorbed in the Peyer's patches ofthe gut and taken up by macrophages in the Peyer's patches. The presenceof liposomal antigen in the Peyer's patches (outside of the macrophages)initiates a local immune response to the antigen as the liposomesbreakdown and release the antigen. The uptake of sufficient liposomalantigen in the macrophages stimulates a systemic immune response, andpreferably a long-term systemic immune response, to the antigen(s) asthe liposomes breakdown within the macrophages to release antigen.

As used herein, “local immune response” refers to mucosal IgA, whichconfers protection from organisms in the bowel lumen and ischaracterized by secretion of local sIgA.

As used herein, “systemic immune response” refers to whole bodyproduction and circulation of organism specific humoral and cellularimmune cells and is characterized by organism specific immune globulin(antibodies) and cytotoxic mononuclear cells.

As used herein, “long term systemic immune response” means a detectiblesystemic immune response to an antigen that lasts at least 150 daysafter administration of the antigen.

As used herein, “sufficient liposomal antigen to stimulate a systemicimmune (or long-term systemic immune) response” means that amount ofantigen-containing liposomes that affect a detectible systemic immuneresponse (or long-term systemic immune response). A systemic immuneresponse may be confirmed by neutralizing antibody testing or othermeans of specific antibody testing, cytotoxic mononuclear cell assaysand in vivo microbe challenge experiments, as is well known in the art.

As used herein, “antigens” may be any substance that, when introducedinto a mammal, will induce a detectable immune response, both humoraland cellular. As used herein, the term “antigen” also includes anyportion of an antigen, e.g., the epitope, which can induce an immuneresponse. In a particularly preferred embodiment of the invention, theantigen is an attenuated or killed microorganism, such as a virus orbacteria, rendering the preparation an oral vaccine against thatmicroorganism.

As used herein, “inactivated HIV I and HIV II antigens” include anysubstance that, when introduced into a mammal, will induce a detectableimmune response, both humoral and cellular. Typical HIV I and HIV IIantigens include but are not limited to, p24 antigen, gp120, gp41, andenvelope proteins.

The liposomes of the present invention may be made of any suitablephospholipid, glycolipid, derived lipid, and the like. Examples ofsuitable phospholipids include phosphatide choline, phosphatidyl serine,phosphatidic acid, phosphatidyl glycerin, phosphatidyl ethanolamine,phosphatidyl inositol, sphingomyelin, dicetyl phosphate,lysophosphatidyl choline and mixtures thereof, such as soybeanphospholipids, and egg yolk phospholipids. Suitable glycolipids includecerebroside, sulphur-containing lipids, ganglioside and the like.Suitable derived lipids include cholic acid, deoxycholic acid, and thelike. The presently preferred lipid for forming the liposomes is eggphosphatidylcholine.

The liposomes may be formed by any of the known methods for formingliposomes and may be loaded with antigen according to known procedures.Known methods for forming liposomal antigen are described, for example,in U.S. Pat. No. 4,235,871 to Papahadjopoulos, et al., and OralMicrobiology and Immunology, 1994, 9:146-153, the disclosures of whichare incorporated herein by reference. What is formed is an emulsioncomprising liposomal antigen.

Viral, bacterial and parasitic antigens may all be incorporated intoliposomes and generate long-term immunity. In all cases, varying thesize of the liposome for each antigen is crucial. The antigens may firstbe individually incorporated into liposomes and then given individuallyor mixed with liposomes containing other antigens. Viral, bacterialand/or parasitic antigens may be combined. In an exemplary embodiment ofthe invention, the liposomes are loaded with p24 antigens. The liposomesmay also be loaded with other HIV antigens or whole virus.

It is also understood that rather than loading multiple viral antigensinto each liposome, preparations may be prepared comprising a mixture ofliposomes wherein each liposome contains only a single antigen. Ifdesired, the liposomes may be loaded with a therapeutic drug in additionto the antigen.

It is preferred that the liposomes used in the present invention have anaverage mean diameter from about 20 nm to about 20 microns, preferablyfrom about 200 nm to about 10 microns, and more preferably of from about1 micron to about 5 microns.

Liposomes larger than about 20 microns are generally not preferredbecause they tend not to be taken up by the macrophages and only affecta local secretory antibody response. That is, the presence of largeantigen-containing liposomes in the lymphoid tissue of the Peyer'spatches will induce gut-associated lymphoid tissue (GALT) to produce IgAantibodies to destroy the antigen. However, no systemic immune responseis induced.

Liposomes smaller than about 20 nm are generally not preferred becausethey also tend not to be processed adequately by macrophages. Thesesmaller liposomes tend to reside in the lymphoid tissue until theyeventually are absorbed into the bloodstream and are destroyed by thereticuloendothelial (RE) system. The smaller liposomes may induce a lowgrade production of secretory IgA, but do not stimulate systemicimmunity.

It has been found that antigen-containing liposomes of from about 20 nmto about 20 microns, preferably from about 200 nm to about 10 micronsand more preferably from about 1 micron to 5 microns tend to be absorbedby macrophages in the Peyer's patches. The macrophages digest theliposomes to release the antigen, which is then presented or displayedat the surface of the macrophage. The macrophages act asantigen-presenting cells which process and present the antigen tosystemic lymphocytes thereby inducing a systemic immune response to theantigen. The macrophages display the antigen in conjunction with themajor histocompatibility complex II (MHC II) glycoproteins to T-helpercells. T-helper cells activate B cells, which proliferate anddifferentiate into mature plasma cells that secrete copious amounts ofimmunoglobulins. In the systemic response, the immunoglobulins secretedare initially IgM followed by IgG.

It is preferred that the liposomes be a mixture of sizes. Suchheterogeneous sizes of liposomes are preferred as they are broken downover a period of time, e.g., up to 180 days or more by the macrophages.Preferably, the mixture of sizes will include liposomes having a size ofabout 20 nm to about 1 micron (small liposomes), liposomes having a sizeof about 1 micron to about 3 microns (medium liposomes) and liposomeshaving a size of about 3 to about 20 microns (large liposomes).Preferred large liposomes are those having a size of from about 3 toabout 5 microns. Preferably, there is at least about 5% by volume ofeach size of liposomes, i.e., small, medium and large, in thecomposition. More preferably, there is at least about 5% by volume ofsmall liposomes, at least 10% by volume medium liposomes, and at least20% by volume large liposomes. A particularly preferred compositioncomprises about 10% by volume small liposomes, about 25% by volumemedium liposomes and about 65% by volume large liposomes.

In a composition containing a heterogeneous population of liposomes,there may be a uniform distribution of sizes or two or more discrete,homogeneous populations. A combination of small, medium and large sizesis preferred because a smoother amnestic antibody curve is generatedproducing the most effective and dependable long-term immunity.

Compositions comprising liposomes of various sizes allow antigens to bereleased in the macrophages over a long period of time, therebycontinuing to stimulate a systemic immune response over a period oftime. The small size liposomes are taken up by the macrophages quicklyand provide an immediate systemic immune response. Medium size liposomesare taken up by the macrophages, but at a slower pace. These liposomesact as a booster, i.e., provide an amnestic response. The larger sizeliposomes take even longer to be taken up by the macrophages and act asa second booster, i.e., provide a second amnestic response. Hence, useof liposomes of varying sizes enables a single dose of theantigen-containing liposomes to be sufficient to result in long term,and even permanent, immunity to the antigen.

The liposomes may be unilamellar or multilamellar. Production ofunilamellar and multilamellar liposomes is also well known in the artand is described, for example, in U.S. Pat. No. 5,008,050 to Cullis etal. and U.S. Pat. Nos. 5,030,453 and 9,522,803 both to Lenk, et al., thedisclosures of which are incorporated herein by reference.

Preparation of a homogeneous population may be accomplished byconventional techniques such as extrusion through a filter, preferablyof 200 nm to 20 micron pore size, the filter being either the straightpath or tortuous path type. Other methods of treating liposomes to forma homogenous size distribution are ultrasonic exposure, the French presstechnique, hydrodynamic shearing, homogenization using, for example, acolloid mill or Gaulin homogenizer, and microfluidization techniques.Microfluidization is one presently preferred method. Other techniquesinvolving sonication are also preferred.

Microfluidization is described, for example, in U.S. Pat. No. 4,533,254to Cook, et al., which is incorporated herein by reference. In apreferred microfluidization procedure, the liposomal emulsion is forcedat high pressure through a small diameter opening and splattered onto awall and then collected.

In a particularly preferred embodiment of the invention, the liposomesare passed one to ten and preferably 4 times through an M-110 SeriesLaboratory Microfluidizer manufactured by Microfluidics Corporation at apressure of, e.g., 14,000 pounds per square inch to achieve a generallyhomogenous population of liposomes having an average mean diameter ofabout 1 micron. Liposomes of other sizes can be prepared using the samemethod by adjusting the number of runs through the microfluidizer, thepressure, and flow rate.

In sonication techniques, the raw materials for the liposomes, e.g.,phospholipids, are combined with antigens, placed in a sonicator, andsonicated for a time, at a temperature and at a speed sufficient toobtain liposomes of the desired size. For example, in a particularlypreferred method, raw materials are placed in a Brinkman Inc. or BeckmanInc. Sonicator and sonicated at 1,000 to 10,000 meters per second at 50°C. for 20, 5 and 2 minutes to obtain small, medium and large liposomes,respectively. Typically, larger sonication times result in smallerliposomes.

To stabilize the liposomal antigen, the emulsion is lyophilized.Lyophilized liposomal antigen can be stored at room temperature for onehalf to three years without degradation of the liposomes or antigen.

Lyophilization may be accomplished by any method known in the art. Suchprocedures are disclosed, for example, in U.S. Pat. No. 4,880,836 toJanoff, et al., the disclosure of which is incorporated herein byreference. Lyophilization procedures preferably include the addition ofa drying protectant to the liposome suspension. The drying protectantstabilizes the liposome suspension. The drying protectant stabilizes theliposomes so that the size and content are maintained during the dryingprocedure and through rehydration. Preferred drying agents aresaccharide sugars including dextrose, sucrose, maltose, manose,galactose, raffinose, trehalose lactose, and triose sugars which arepreferably added in amounts of about 5% to about 20% and preferablyabout 10% by weight of the aqueous phase of the liposomal suspension.Dextrose, sucrose and maltose are presently preferred. Manitol may beused in conjunction with any of the saccharides. Additionalpreservatives such as BHT or EDTA, urea, albumin, dextran or polyvinylalcohol may also be used.

The lyophilized liposomal antigen may be packaged for oraladministration in either a pill form or a capsule. An enteric coating ispreferably applied to the liposomal antigen to prevent breakdown in thestomach.

The enteric coating may be made of any suitable composition. Suitableenteric coatings are described, for example, in U.S. Pat. Nos. 4,311,833to Namikoshi, et al.; 4,377,568 to Chopra; 4,385,078 to Onda, et al.;4,457,907 to Porter; 4,462,839 to McGinley, et al.; 4,518,433 toMcGinley, et al.; 4,556,552 to Porter, et al.; 4,606,909 to Bechgaard,et al.; 4,615,885 to Nakagame, et al.; and 4,670,287 to Tsuji, all ofwhich are incorporated herein by reference.

Preferred enteric coating compositions include alkyl and hydroxyalkylcelluloses and their aliphatic esters, e.g., methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,hydroxybutylcellulose, hydroxyethylethylcellulose,hydroxyprophymethylcellulose, hydroxybutylmethylcellulose,hydroxypropylcellulose phthalate, hydroxypropylmethylcellulose phthalateand hydroxypropylmethylcellulose acetate succinate;carboxyalkylcelluloses and their salts, e.g.,carboxymethylethylcellulose; cellulose acetate phthalate;polycarboxymethylene and its salts and derivatives; polyvinylalcohol andits esters, polycarboxymethylene copolymer with sodium formaldehydecarboxylate; acrylic polymers and copolymers, e.g., methacrylicacid-methyl methacrylic acid copolymer and methacrylic acid-methylacrylate copolymer; edible oils such as peanut oil, palm oil, olive oiland hydrogenated vegetable oils; polyvinylpyrrolidone;polyethyleneglycol and its esters, e.g., and natural products such asshellac.

Other preferred enteric coatings include polyvinylacetate esters, e.g.,polyvinyl acetate phthalate; alkyleneglycolether esters of copolymerssuch as partial ethylene glycol monomethylether ester ofethylacrylate-maleic anhydride copolymer or diethyleneglycolmonomethylether ester of methylacrylate-maleic anhydride copolymer,N-butylacrylate-maleic anhydride copolymer, isobutylacrylate-maleicanhydride copolymer or ethylacrylate-maleic anhydride copolymer; andpolypeptides resistant to degradation in the gastric environment, e.g.,polyarginine and polylysine.

Mixtures of two or more of the above compounds may be used as desired.

The enteric coating material may be mixed with various excipientsincluding plasticizers such as triethyl citrate, acetyl triethylcitrate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, dibutyltartrate, dibutyl maleate, dibutyl succinate and diethyl succinate andinert fillers such as chalk or pigments.

The composition and thickness of the enteric coating may be selected todissolve immediately upon contact with the digestive juice of theintestine. Alternatively, the composition and thickness of the entericcoating may be selected to be a time-release coating which dissolvesover a selected period of time, as is well known in the art.

EXAMPLE 1

To establish the effective absorption of lyophilized liposomes byPeyer's patches and uptake by macrophages, the following protocol wasfollowed:

Preparation of antigen-containing liposomes:

Antigen-containing liposomes having a diameter of approximately 142nanometers used in the experimental study described below were preparedaccording to the following procedure.

1. 2250 ml of water (double distilled) to beaker (keep cool) and setwith a nitrogen sparge for at least 30 minutes.

2. Add 225 gms of maltose (Sigma M5885) to the water and mix untildissolved. Keep the nitrogen sparge going. Mixture at ph of 4.81.

3. In another beaker 10.59 gms of egg phosphatidylcholine (EPC) (Sigma)is combined with 8.38 ml of ethanol (anhydrous, Sigma E3884) and mixeduntil dissolved. To this add 67.5 mg of BHT and mix until dissolved. Tothis mixture add 2160 mg of purified Coxsackie B viral antigen and mixuntil dissolved. Use the remaining 4.19 ml of ethanol to rinse anyremaining Coxsackie B antigen in the weighing container into themixture.

4. Draw the ethanol solution into a 10 ml glass syringe and add to themaltose solution over 11 minutes with continued nitrogen sparge. Keep ph<7.0 (goes into microfluidizer at ph 4.81). Measure. Hand blade mixture.Keep everything cool, e.g. 1.5 degrees C.

5. Microfluidizer. Four (4) passes through the microfluidizer at 110°F.:

Weight of Materials to be Used EPC 10.59 grams Maltose 225 grams Ethanol12.57 ml BHT 67.5 mg Coxsackie B 2160 mg (USP) Water 2250 ml Pressure16,000 PSI

6. Take 2.7 ml of the finished product and lyophilize in approximately1,000 6 ml Wheaton eye dropper bottles. Lyophilization was accomplishedaccording to the following cycle:

1. Shelfat ≦−45° C. for at least one (1) hour before loading.

2. Load product keep at ≦−45° C. for twelve (12) hours.

3. Vacuum to ˜50μ.

4. Shelf temperature at −28° C. to −20° C. for 59 hours.

5. Shelf temperature rose from −20° C. to −5° C. during subsequent ten(10) hours. Visually product needed extra time at −20° C.

6. Shelf reset at −22° C. and maintained at −22° C. to −18° C. forthirty-six (36) hours.

7. Shelf reset +25° C. and held at 25° C. for 48 hours.

It is anticipated that the following lyophilization cycle will providethe same results in a shorter time.

1. Shelf to ≦45 ° C. for at least one (1) hour before loading.

2. Load product, keep at ≦−45° C. for at least six (6) hours.

3. Vacuum to ≦100μ.

4. Shelf to −28° C. for 50 hours.

5. Shelf to +25 ° C. for 40-50 hours.

Experimental:

100 micrograms of lyophilized liposomes 142 nanometers in diameter weresuspended in 0.3 ml of 0.5% xanthum gum aqueous solution. The mixturewas given via a gavage tube to four week old male CD-1 mice. Five micewere given the liposomal preparation and five mice were given 0.5%xanthum only as controls. For one week the ten mice were kept on ad libdiet and water ad lib. On day seven the mice were anesthetized withmethyloxyfluorane and through a mid-line abdominal incision theperitoneum was entered. The small bowel was resected and examined forthe Peyer's patches. The Peyer's patches from the small bowel wereremoved and placed in one molar phosphate buffer minced with a straightrazor into less than 1 mm sections on wax paper.

The preparation was then fixed at room temperature with 4%glutaraldehyde in two molar phosphate buffer, washed three times withone molar phosphate buffer and taken to the electronmicroscopy facility.The preparation was then dehydrated and mounted in epoxy resin, cut witha microtome, stained with osmium tetroxide, then examined under a ZeisCR10 electron microscope. The Peyer's patches were then photographed andlabeled as noted.

The spleen and Peyer's patches of the gut were sectioned and slides wereprepared. Photomicrographs were taken and are presented here as FIGS.1-12. The photomicrographs show liposomes (FIG. 1) residing in venulesand extracellular tissue of the Peyer's patch (FIGS. 2, 6, 7, 8, 12).They also show that the liposomes were not present in the spleniclymphoid tissue which indicate that the liposomes were staying in thePeyer's patches and not circulating through the blood stream in themouse. (FIGS. 3, 4). Finally, the photomicrographs show liposomes beingabsorbed and digested by macrophages (FIGS. 5, 9, 10, 11).

EXAMPLE 2

Virus and Cells

Virus stocks of CVB5 strain C59 were prepared in monolayers of monkeykidney (MK) cells using an inoculum giving an MOI of 1 pfu/cell insupplemental Leibovitz's L15 medium as described in See D M Tilles J G.,“EFfficacy of a Polyvalent Inactivated-virus Vaccine in Protecting Micefrom Infection with Clinical Strains of Group B Coxsackie viruses.”Scand. J. Infect. Dis. 26:739-747, 1994, the disclosure of which isincorporated herein by reference. Flasks were observed daily forcytopathic effect (cpe) until cpe reached 4+. At that time, the viruswas harvested, aliquoted and frozen at −80° C. until further use.

Animals

Male CD-1 Mice 16-18 g were obtained from Charles Rivers Farms,Wilmington, Mass.

Preparation of Viral Antigen

One strain of coxsackieviruses groups B1-6 were absorbed to monolayersof MK cells at a multiplicity of 1 pfu/cell and incubated as described.When maximal cytopatic effect was observed, the virus-containing mediafor a single strain was harvested and pooled. Aliquots were stored andtested for viral titer as previously described in See, D. M., Tilles, J.G., “Efficacy of a Polyvalent Inactivated-virus Vaccine in ProtectingMice from Infection with Clinical Strains of Group B Coxsackieviruses.”Scand. J. Infect. Dis. 26:739-747, 1994.

Microencapsulation

Viral proteins were encapsulated with 3 different particle sizeliposomes as follows: Before beginning, 3 round bottom flasks werelabeled A (2 minutes), B (5 minutes) and C (20 minutes). 783 mg ofdiphosphatidylchoxene (DPPC) (Avanti), 180 mg Cholesterol (Sigma), and36mg Dicetyl-Phosphate (Sigma) was added to each of the flasks. 25 mgN-(1-pyrene sulfonyl)-1,2 hexadecanoyl-sn-glycero-3 phosphoethanolamine,triethylammonium sale (PS DHPE) (Molecular Probes Inc.) was thendissolved in 1 ml of chloroform and 320 ul (8 mg) was added into each ofthe round bottom flasks. Next, 2680 (3 ml-320 ul) of chloroform wasadded to each flask. Each flask was then placed in a rotovapor(Brinkman) with water bath set to 45° C. until dry. All availableantigen was pooled into a 200 ml beaker in a hoop equipped with a Hepafilter and mixed well. 250 mg of maltose (Sigma) was measured into 3 50ml centrifuge tubes. 50 ml of pooled antigen containing about 4×10⁵ pfuof each virus was added to each tube and then one tube was added toflask C and warmed for 5 min in 50° C. water bath and then sonicated ina sonicator manufactured by Brinkman Inc. at a setting of 10,000 metersper second adjustable to 1-100,000 meters per second and at the sametemperature for 20 minutes. The second tube of antigen/maltose mixturewas added to flask B, warmed for 5 minutes in 50° C. water bath andsonicated for 5 minutes. The last tube of antigen/maltose was added toflask A, warmed for 5 minutes in 50° C. water bath and sonicated for 2minutes. Aliquots of 1 ml were then removed for particle sizing. Theremaining batches were placed in separate specimen cups labeledappropriately and placed at −70° C. until lyophilization.

Immunizations and Experimental Methods

The resultant 3 liposomes (2 um, 10 um, 908 nm) were given to male CD-1mice weighing 16-18 g obtained from Charles Rivers Farms, Wilmington,Mass. orally either alone or mixed for either 1 2 or 3 doses over thesame number of weeks. For all experiments, 30 mg liposomes were givenorally in 0.3 cc containing sodium acetate buffer, pH 9.0. In oneexperiment, 120 mg of mixed liposomes were given. The final set of micewere given either mixed liposomes or a placebo and then infected withCVB5/C59.

The mice were then sacrificed. Blood samples, Peyer's patches andspleens were taken for microtiter neutralization antibody titrationassays and Electron Microscopy work respectively. Pancreas samples weretaken only from infected mice to run viral titer assays.

Neutralizing Antibody Titration Assay

For each mouse, a serum sample was taken, prepared, and assayed forantibody response as previously described in See, D. M., Tilles, J. G.,“Efficacy of a Polyvalent Inactivated-virus Vaccine in Protecting Micefrom infection with Clinical Strains of Group B Coxsackieviruses,”Scand. J. Infect. Dis. 26:739-747, 1994. After serum and virus wereincubated at room temperature for 1 hour, MK cells from one 75 cm²tissue flask were added directly to the microtiter plate.

Virus Assay

For each mouse, a pancreas sample was taken, homogenized in supplementedL15 diluent and assayed for virus by the plaque technique describedpreviously in See, D. M., Tilles, J. G., “Treatment of Coxsackievirus A9Myocarditis in Mice with WIN 54954,” Antimicrob. Agents Chemother.36:425-428, 1992, the disclosure of which is incorporated herein byreference, with the modification of using MK rather than ForeskinFibroblast cells.

Electron Microscopy

Peyer's patches and spleens were diced into pieces <1 mm with a singleedged blade on a wax sheet and kept moist in 0.1M phosphate buffer. Thepieces were then added to a vial of gluteraldehyde solution prepared bymixing 0.2M phosphate buffer, pH 7 (28 ml 0.2M NaH₂PO₄+72 ml 0.2MNa₂HPO₄) 1:1 with 8% gluteraldehyde (Ted Pella Inc.). Tissue was fixed2-4 hours at room temperature and then washed 3 times with 0.1Mphosphate buffer. Samples were then taken to University of CaliforniaIrvine Imaging Facility to process for the Electron Microscopy.

Results

Induction of Antibody

To show the success of the liposome vaccine in stimulating a specificantibody response in mice, serial determinations of neutralizingantibody to all six coxsackie B serogroups were made in groups of 5 micefor each liposome tested. Means for each liposome were calculated forneutralizing antibody titer in plasma obtained 8-60 days after finaldose of vaccine. Eight days after final dose of liposomes, a modest risein titer to all strains tested was recorded. The smallest liposome (909nm) gave the largest initial response after one dose (mean 4.2+/−SD2.3)but had little increase with repeated doses. The largest (10 um)liposome resulted in the greatest antibody response after 3 doses butdid not result in detectable antibody levels 24 days after final dose. Asingle dose of the mixed liposomes produced an antibody response stilldetectable 21 days after final dose.

The results are shown in Table 1 below.

TABLE 1 Neutralizing antibody titers to 6 CVB strains after variousdoses of vaccine. Days since Mean Neutralizing Liposomes # doses lastdose Antibody Titer 980 nm 1 8 4.2+/−2.3 2 8 4.9+/−2.5 3 8 4.7+/−2.7  2um 1 8 3.3+/−1.8 2 8 4.7+/−2.9 3 8 6.2+/−3.6  10 um 1 8 3.1+/−1.5 2 83.8+/−1.9 3 8 6.9+/−3.3 1 24 <3 Mixed 1 8 3.9+/−1.7 2 8 4.9+/−2.5 3 87.6+/−2.9  1* 24 4.8+/−2.7  3* 24 8.8+/−3.3   1+ 60 4.7+/−2.9 Notes: n =5 for each group. Titers <3 were assigned a value of 2 for purposes ofdetermining the mean. Means are for all 6 coxsackie B serogroups. *30 mg+120 mg

Protection from Acute Infection with CVB5/C59 Viral Titer Assay

To confirm the ability of the oral vaccination to limit challenge virusinfection, titers of virus were determined in the pancreas of micekilled 3 days after infection. Two groups of 5 mice were used; one groupwas given 3 doses of mixed liposomes and the other group was givenbuffer placebo. The placebo group ended up with a mean titer of 5.3×10⁴(pfu/mg) while the vaccine group's mean titer was only 2.2×10² (pfu/mg).(Titers of <2 (the lower limit of sensitivity of the assay) wereassigned a value of 1 for the purpose of calculating the mean.)

Electron Microscopy

As shown in FIG. 13, seven days after final oral inoculation, 980 nmliposomes are visible in vacuoles within macrophages of the Peyer'sPatches. As shown in FIG. 14, 21 days after oral inoculation, a 10micron liposome was observed in a macrophage of the Peyer's patches. Asshown in FIG. 15, 60 days after oral inoculation, 2 micron liposomeswere observed in a vacuole within a macrophage of the Peyer's Patches.

EXAMPLE 3

Antigen

p24 antigen was purchased from Biodesign International, Kennebunk, Me.

Animals

Male CD-1 Mice 16-18 g were obtained from Charles Rivers Farms,Wilmington, Mass.

Microencapsulation

p24 antigen was encapsulated in 3 different particle size liposomes asfollows: A solution of PyS DHPE was prepared in a test tube bydissolving 25 mg of PyS DHPE in 1.0 ml, of chloroform. Lipid solutionwas then prepared in a separate test tube by combining 313 mg of DPPC,72 mg of cholesterol, 14 mg of dicetylphosphate, 144 μL PyS DHPEsolution, and 1.056 ml of chloroform, for a total volume ofapproximately 1.20 mL.

900 μL, of the lipid solution was aliquoted into a glass test tube. Thesolvent in each tube was evaporated to dryness with Nitrogen gas. Amaltose solution was prepared by dissolving 100 mg of maltose in 1.0 mLof water. 750 μL of maltose solution was measured into the three testtubes. 200 μL of p24 antigen was added to each tube, then 4550 μL ofwater was added.

The solution in the first tube was warmed at 50° C. for 2 minutes thensonicated at 50° C. for 2 minutes to obtain liposomes have a diameter ofapproximately 5 μm. The solution in the second tube was warmed at 50° C.for 2 minutes then sonicated at 50° C. for 5 minutes to obtain liposomeshaving a diameter of approximately 2 μm. The solution in the third testtube was warmed at 50° C. for 2 minutes, giving liposomes having a sizeof approximately 5 μm. Approximately 10 μl of liposomes was removed fromeach test tube for particle sizing. The liposomes from all three testtubes were combined for a total volume of approximately 15 ml. Thesolution was aliquoted into glass vials (1.5 mL/vial; 60 μg/vial). Thevials were placed in a −10° C. freezer overnight. The samples were thenlyophilized.

Immunizations and Experimental Methods

The resultant lyophilized liposome mixtures of three different-sizedliposomes (1 μm, 2 μm, and 5 μm) were orally administered to male CD-1mice weighing 16-18 g obtained from Charles Rivers Farms, Wilmington,Mass. For all experiments, 30 mg liposome mixtures were orallyadministered in 0.3 cc containing sodium acetate buffer, pH 9.0, so thateach mouse received 60 μg of p24 antigen. The mice were then sacrificed.Blood samples, Peyer's patches and spleens were taken for antibody top24 antigen by Enzyme Immunolinked Assay (EIA), Electron Microscopywork, and lymphocyte proliferation assay, respectively.

Electron Microscopy

Peyer's patches and spleens were diced into pieces <1 mm with a singleedged blade on a wax sheet and kept moist in 0.1M phosphate buffer. Thepieces were then added to a vial of gluteraldehyde solution prepared bymixing 0.2M phosphate buffer, pH 7 (28 ml 0.2M NaH₂PO₄+72 ml 0.2MNa₂HPO₄) 1:1 with 8% gluteraldehyde (Ted Pella Inc.). Tissue was fixed2-4 hours at room temperature and then washed 3 times with 0.1Mphosphate buffer. Samples were then taken to University of CaliforniaIrvine Imaging Facility to process for the Electron Microscopy.

Induction of Antibody

To show the success of the liposome vaccine in stimulating a specificantibody response in mice, EIA assays to p24 antigen were conducted atweekly intervals for 5 weeks. By the fifth week, both mice assayed haddeveloped antibodies to the p24 antigen.

Lymphocyte Proliferation Assay

To determine the ability of liposomal p24 antigen to induce a cellularimmune response, a lymphocyte proliferation assay was performed afterweek two and week four. Proliferation of splenic mononuclear cells wassignificantly enhanced in liposomal p24 antigen-treated mice compared tountreated control mice, as shown in Table 2 below. The proliferationindex indicates the extent of cellular proliferation resulting fromprior exposure to p24 antigen compared to a control not previouslyexposed to the antigen, and takes into account the amount of p24 antigenadded to the assay. A higher proliferation index indicates more cellularproliferation and therefore a better cellular immune response. Aproliferation index of at least 1 indicates a very active immuneresponse.

TABLE 2 Added Antigen Proliferation Index Week 2 40 mcg 4.95 80 mcg 6.16120 mcg  5.58 Week 4 0.1 mcg  <1.0  1 mcg 1.5 10 mcg 4.3

Electron Microscopy

By the second week after vaccination, liposomes were seen in macrophagesof the Peyer's patches.

The preceding description has been presented with reference to presentlypreferred embodiments of the invention. Workers skilled in the art andtechnology to which this invention pertains will appreciate thatalterations and changes in the described structure may be practicedwithout meaningfully departing from the principal, spirit and scope ofthis invention.

Accordingly, the foregoing description should not be read as pertainingonly to the precise embodiments described and illustrated in theaccompanying drawings, but rather should be read consistent with and assupport to the following claims which are to have their fullest and fairscope.

What is claimed is:
 1. A method for stimulating a systemic immuneresponse to an antigen selected from the group consisting of inactivatedHIV I and HIV II antigens and combinations thereof in a mammalcomprising: providing a lipsomal preparation comprising lyophilizedliposomes containing at least one antigen selected from the groupconsisting of inactivated HIV I and HIV II antigens, wherein theliposomes have at least three different sizes and consist essentiallyof: at least 5% by volume, based on the total volume of liposomes in thepreparation, small liposomes having a size, before lyophilization, offrom about 20 nm to about 1 micron, at least 5% by volume, based on thetotal volume of liposomes in the preparation, medium liposomes having asize, before lyophilization, of from about 1 micron to about 3 microns,and at least 5% by volume, based on the total volume of liposomes in thepreparation, large liposomes having a size, before lyophilization, offrom about 3 microns to about 20 microns; and orally administering aneffective amount of the liposomal preparation to a mammal, wherebysufficient antigen containing liposomes are absorbed in the Peyer'spatches of the gut of the mammal and are taken up by macrophages in thePeyer's patches to stimulate a systemic immune response.
 2. A method asclaimed in claim 1, wherein the liposomes are multi-lamellar beforelyophilization.
 3. A method as claimed in claim 1, wherein the liposomalpreparation is contained within an enterically-coated capsule.
 4. Amethod as claimed in claim 1 wherein the liposomes consist essentiallyof at least 5% by volume, based on the total volume of liposomes in thepreparation, small liposomes having a size, before lyophilization, offrom about 20 nm to about 1 micron, at least 10% by volume, based on thetotal volume of liposomes in the preparation, medium liposomes having asize, before lyophilization, of from about 1 micron to about 3 microns,and at least 20% by volume, based on the total volume of liposomes inthe preparation, large liposomes having a size, before lyphilization, offrom about 3 microns to about 20 microns.
 5. A method as claimed inclaim 1 wherein the liposomes comprise about 10% by volume, based on thetotal volume of liposomes in the preparation, small liposomes having asize, before lyophilization, of from about 20 nm to about 1 micron,about 25% by volume, based on the total volume of liposomes in thepreparation, medium liposomes having a size, before lyophilization, offrom about 1 micron to about 3 microns, and about 65% by volume, basedon the total volume of liposomes in the preparation, large liposomeshaving a size, before lyopilization, of from about 3 microns to about 20microns.
 6. A method as claimed in claim 1 wherein the liposomescomprise at least two different antigens.
 7. A preparation for oraladministration to a mammal capable of stimulating a systemic immuneresponse to at least one antigen selected from the group consisting ofinactivated HIV I and HIV II antigens, said preparation comprising aneffective amount of lyophilized antigen-containing liposomes, whereinthe liposomes have at least three different sizes, beforelyophilization, and consist essentially of: at least 5% by volume, basedon the total volume of liposomes in the preparation, small liposomeshaving a size, before lyophilization, of from about 20 nm to about 1micron, at least 5% by volume, based on the total volume of liposomes inthe preparation, medium liposomes having a size, before lyophilization,of from about 1 micron to about 3 microns, and at least 5% by volume,based on the total volume of liposomes in the preparation, largeliposomes having a size, before lyphilization, of from about 3 micronsto about 20 microns.
 8. A preparation as claimed in claim 7 wherein theliposomes are multi-lamellar before lyophilization.
 9. A preparation asclaimed in claim 7 wherein the liposome preparation is contained withinan enterically-coated capsule.
 10. A preparation as claimed in claim 7wherein the liposomes comprise small, medium and large liposomes.
 11. Apreparation as claimed in claim 7 wherein the liposomes consistessentially of at least 5% by volume, based on the total volume ofliposomes in the preparation, small liposomes having a size, beforelyophilization, of from about 20 nm to about 1 micron, at least 10% byvolume, based on the total of liposomes in the preparation, mediumliposomes having a size, before lyophilization, of from about 1 micronto about 3 microns, and at least 20% by volume, based on the totalvolume of liposomes in the preparation, large liposomes having a size,before lyophilization, of from about 3 microns to about 20 microns. 12.A preparation as claimed in claim 7 wherein the liposomes consistessentially of about 10% by volume, based on the total volume ofliposomes in the prepartion, small liposomes having a size, beforelyophilization, of from about 20 nm to about 1 micron, about 25% byvolume, based on the total volume of liposomes in the preparation,medium liposomes having a size, before lyophilization, of from about 1micron to about 3 microns, and about 65% by volume, based on the totalvolume of liposomes in the preparation, large liposomes having a size,before lyophilization, of from about 3 microns to about 20 microns. 13.A preparation as claimed in claim 7 wherein the liposomes comprise atleast two different antigens.
 14. A method according to claim 1, whereinthe large liposomes have a size, before lyophilization, of from about 3microns to about 10 microns.
 15. A method according to claim 1, whereinthe large liposomes have a size, before lyophilization, of from about 3microns to about 5 microns.
 16. A method according to claim 1, whereinthe small liposomes have a size, before lyophilization, of about 1micron.
 17. A method according to claim 1, wherein the medium liposomeshave a size, before lyophilization, of about 2 microns.
 18. A methodaccording to claim 1, wherein the large liposomes have a size, beforelyophilization, of about 10 microns.
 19. A method according to claim 1,wherein the small liposomes have a size, before lyophilization, of about1 micron, the medium liposomes have a size, before lyophilization, ofabout 2 microns, and the large liposomes have a size, beforelyophilization, of about 10 microns.
 20. A method according to claim 4,wherein the large liposomes have a size, before lyophilization, of fromabout 3 microns to about 10 microns.
 21. A method according to claim 4wherein the large liposomes have a size, before lyophilization, of fromabout 3 microns to about 5 microns.
 22. A method according to claim 4,wherein the small liposomes have a size, before lyophilization, of about1 micron.
 23. A method according to claim 4, wherein the mediumliposomes have a size, before lyophilization, of about 2 microns.
 24. Amethod according to claim 4, wherein the large liposomes have a size,before lyophilization, of about 10 microns.
 25. A method according toclaim 4, wherein the small liposomes have a size, before lyophilization,of about 1 micron, the medium liposomes have a size, beforelyophilization, of about 2 microns, and the large liposomes have a size,before lyophilization, of about 10 microns.
 26. A method according toclaim 5, wherein the large liposomes have a size, before lyophilization,of from about 3 microns to about 10 microns.
 27. A method according toclaim 5, wherein the large liposomes have a size, before lyphilization,of from about 3 microns to about 5 microns.
 28. A method according toclaim 5, wherein the small liposomes have a size, before liphilization,of about 1 micron.
 29. A method according to claim 5, wherein the mediumliposomes have a size, before lyophilization, of about 2 microns.
 30. Amethod according to claim 5, wherein the large liposomes have a size,before lyophilization, of about 10 microns.
 31. A method according toclaim 5, wherein the small liposomes have a size, before lyophilization,of about 1 micron, the medium liposomes have a size, beforelyophilization, of about 2 microns, and the large liposomes have a size,before lyophilization, of about 10 microns.
 32. A prepairation accordingto claim 7, wherein the large liposomes have a size, beforelyophilization, of from about 3 microns to about 10 microns.
 33. Apreparation according to claim 7, wherein the large liposomes have asize, before lyophilization, of from about 3 microns to about 5 microns.34. A preparation according to claim 7, wherein the small liposomes havea size, before lyophilization, of about 1 micron.
 35. A preparationaccording to claim 7, wherein the medium liposomes have a size, beforelyophilization, of about 2 microns.
 36. A preparation according to claim7, wherein the large liposomes have a size, before lyophilization, ofabout 10 microns.
 37. A preparation according to claim 7, wherein thesmall liposomes have a size, before lyophilization, of about 1 micron,the medium liposomes have a size, before lyophilization, of about 2microns and the large liposomes have a sizes before lyophilization, ofabout 10 microns.
 38. A preparation according to claim 11, wherein thelarge liposomes have a size, before lyophilization, of from about 3microns to about 10 microns.
 39. A preparation according to claim 11,wherein the large liposomes have a size, before lyophilization, of fromabout 3 microns to about 5 microns.
 40. A preparation according to claim11, wherein the small liposomes have a size, before lyophilization, ofabout 1 micron.
 41. A preparation according to claim 11, wherein themedium liposomes have a size, before lyophilization, of about 2 microns.42. A preparation according to claim 11, wherein the large liposomeshave a size, before lyophilization, of about 10 microns.
 43. Apreparation according to claim 11, wherein the small liposomes have asize, before lyophilization, of about 1 micron, the medium liposomeshave a size, before lyophilization, of about 2 microns, and the largeliposomes have a size, before lyophilization, of about 10 microns.
 44. Apreparation according to claim 12, wherein the large liposomes have asize, before lyophilization, of from about 3 microns to about 10microns.
 45. A preparation according to claim 12, wherein the largeliposomes have a size, before lyophilization, of from about 3 microns toabout 5 microns.
 46. A preparation according to claim 12, wherein thesmall liposomes have a size, before lyophilization, of about 1 micron.47. A preparation according to claim 12, wherein the medium liposomeshave a size, before lyophilization, of about 2 microns.
 48. Apreparation according to claim 12, wherein the large liposomes have asize, before lyophilization, of about 10 microns.
 49. A preparationaccording to claim 12, wherein the small liposomes have a size, beforelyophilization, of about 1 micron, the medium liposomes have a size,before lyophilization, of about 2 microns, and the large liposomes havea size, before lyophilization, of about 10 microns.
 50. A methodaccording to claim 1 wherein the antigen containing liposomes arecapable of being absorbed in the Peyer's patches of the gut of themammal and are capable of being taken up by macrophages in the Peyer'spatches to stimulate a systemic immune response without the presence ofan adjuvant.
 51. A method according to claim 1 wherein the antigencontaining liposomes are capable of being absorbed in the Peyer'spatches of the gut of the mammal and are capable of being taken up bymacrophages in the Peyer's patches to stimulate a systemic immuneresponse without generating a typical adjuvant effect.